Memory T cells specific for donor antigens present a unique challenge in transplantation. While memory T cells augment host protection upon re-infection, donor-reactive memory T cells lead to robust immune responses to a transplanted organ translating into increased risk of poor allograft outcome in humans. Furthermore, memory T cells appear to be resistant to the effects of currently used graft-prolonging therapies including immunosuppressive drugs, depletional strategies and conventional costimulatory blockade. The existence of two populations of memory CD4 T cells has been proposed based on distinct migratory properties. While central memory CD4 T cells reside in secondary lymphoid organs, effector memory CD4 T cells preferentially circulate through non-lymphoid peripheral tissues and perform pathogen surveillance. The functions of these memory C04 T cells subsets, their contribution to allograft rejection and susceptibility to the currently used immunotherapies remain unknown and need to be tested. The goal of the proposed studies is to determine how the anatomical location of lymphoid and peripheral alloreactive memory CD4T cells affects their activation, function and susceptibility to lymphoid sequestration and to costimulatory blockade. We hypothesize that lymphoid but not peripheral memory CD4T cells contribute to cardiac allograft rejection by providing help for the induction of donor-reactive CDST cells and alloantibody while peripheral memory CD4T cells migrate into the graft and express functions mediating tissue damage. We further hypothesize that costimulatory ICOS/B7RP-1 interactions are required for helper functions ofmemory CD4 T cells and for the migration of both memoryand newly generated effector T cells into the graft. To test this hypothesis, we propose to comparethe migratory properties and functions of lymphoid versus peripheral memory CD4 T cells, to test the effect of lymphoid sequestration on the function of lymphoidversus peripheral alloreactive memory CD4 T cells, and to determine the role of costimulation through ICOS/B7RP- 1 in the re-activation, trafficking and functions of lymphoid and peripheral memory CD4T cells. The results of the proposed experiments will provide mechanistic insights into the immunobiology of alloreactive memory CD4 T cells in allograft rejection. This information should contribute to the development of combinatorial therapies to improve allograft function and survival in sensitized patients.